One gene, two phenotypes – understanding the pathomechanics and leukemia development in congenital neutropenia and cyclic neutropenia

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Severe congenital neutropenia (CN) is a mono-lineage pre-leukemia bone marrow failure syndrome, characterized by early onset of neutropenia accompanied by severe infections. CN is a heterogeneous disease, caused by mutations in different genes, but most commonly in ELANE, encoding the neutrophil elastase (NE) protein, which is inherited as an autosomal dominant disease. A second form of hereditary neutropenia is cyclic neutropenia (CyN), which is characterized by oscillations of the neutrophil counts with a nadir occurring every 21 days. During the periods of low neutrophil counts, patients may suffer from infections, such as aphthous stomatitis, periodontitis and typhlitis. Almost all cases of CyN result from mutations in ELANE and, remarkably, the mutations causing CyN invariably overlap with those described in CN. It is unclear how the same mutation can cause CN and CyN. One severe complication of CN, and to a much lesser extent of CyN, is the development of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Granulocyte-colony stimulating factor (G-CSF) is a life-saving drug for CN and CyN patients, improving the granulopoiesis and preventing severe infections. However, the risk of leukemia development in CN directly correlates with the G-CSF dose required. The cumulative incidence of MDS or AML in CN is around 20% after 10 years of G-CSF treatment. Patients with CyN have a much lower but existent susceptibility to develop MDS and AML. It is unclear why leukemia progression is much lower in CyN patients. No mouse model exists to study CN and CyN caused by ELANE mutations, as targeting the mouse ortholog of ELANE in mice does not cause neutropenia. In addition, as CN and CyN are often caused by identical mutations in ELANE, it is not possible to generate accurate models that will differentiate between the two disorders by simple gene editing methods. Therefore, we aim to generate a model for CN and CyN caused by ELANE mutations and for the malignant transformation in CN, using patients-derived induced pluripotent stem cells (iPSCs). Importantly, iPSCs carry the full genetic background of the patients, and will therefore include any possible genetic modifiers that have not been identified so far. We recently published the generation and granulocytic differentiation of patients-derived iPSCs, including for CN caused by mutations in ELANE. iPSCs recapitulated the defective granulopoiesis observed clinically. No published study utilized an iPSCs model of CyN. We plan to compare pathomechanics of CN and CyN using patients-derived iPSCs. In addition, as CN patients carry a high risk for leukemic transformation, related to acquired G-CSFR mutations, we aim to generate a model for pre-leukemia by introducing these mutations using CRISPR/Cas9. We believe that this model will help elucidate the pathways involved in early malignant transformation processes in CN and may reveal more general pathways contributing to malignant transformation.